CPT-11 is a camptothecin prodrug that is activated by esterases to yield SN-38, a potent topoisomerase I poison. CPT-11 has been approved for use against colon cancer and is currently being screened for other childhood and adult malignancies. We have recently characterized a series of carboxylesterases (CEs) that demonstrate dramatic differences in their ability to activate CPT-11. This proposal details the structural analyses of these CEs to unravel the role they play in CPT-11 activation. The long-range goal of this project is to use the structures of the enzymes involved in CPT-11 metabolism to improve the efficacy and use of CPT-11. By combining the tools of x-ray crystallography, biochemistry and in vivo studies, we propose to unravel the structural basis of CPT-11 activation. These studies should facilitate the development of novel CPT-11's, the design of CE inhibitors to reduce drug side effects, and the use of CEs in viral-directed cancer co-therapies. The hypothesis to be tested is that subtle structural differences between carboxylesterases play central roles in the activation of CPT-11 in vivo. We will examine three mammalian CEs. The first, a rabbit liver carboxylesterase (rCE), efficiently activates CPT-11. We have determined the crystal structure of the rCE glycoprotein in complex with a product of CPT-11 activation to 2.5 A resolution. This is the first structure of a mammalian CE. Two human carboxylesterases, carboxylesterase 1 (hCE1) and intestinal carboxylesterase (hiCE), will also be examined, hCE1 is similar in sequence torCE (81 percent identity) but does not activate CPT-11. hiCE, in contrast, shares only 47 percent sequence identity with rCE but does efficiently activate CPT-11. Unraveling the structural basis of these differences is a central focus of this proposal. Five specific aims will be pursued combining the tools of x-ray crystallography with biochemical and in vivo studies: 1. Elucidate how CPT-11 binds to the rabbit liver CE and unravel the mechanism of drug activation. 2. Determine why human CE1 is unable to activate CPT-11 despite its high sequence similarity with rCE. 3. Examine the crystal structure of human intestinal CE to illuminate why this enzyme efficiently activates CPT-11. 4. Characterize, both structurally and functionally, mutants of rCE, hCE1 and hiCE designed to establish the molecular determinants of CPT-11 activation. 5. Assess the efficacy of drug activation and the ability to sensitize cells expressing mutant forms of rCE, hCE1 and hiCE to CPT-11.